Robust RNAi-based resistance to mixed infection of three viruses in soybean plants expressing separate short hairpins from a single transgene

Transgenic plants expressing double-stranded RNA (dsRNA) of virus origin have been previously shown to confer resistance to virus infections through the highly conserved RNA-targeting process termed RNA silencing or RNA interference (RNAi). In this study we applied this strategy to soybean plants and achieved robust resistance to multiple viruses with a single dsRNA-expressing transgene. Unlike previous reports that relied on the expression of one long inverted repeat (IR) combining sequences of several viruses, our improved strategy utilized a transgene designed to express several shorter IRs. Each of these short IRs contains highly conserved sequences of one virus, forming dsRNA of less than 150 bp. These short dsRNA stems were interspersed with single-stranded sequences to prevent homologous recombination during the transgene assembly process. Three such short IRs with sequences of unrelated soybean-infecting viruses (Alfalfa mosaic virus, Bean pod mottle virus, and Soybean mosaic virus) were assembled into a single transgene under control of the 35S promoter and terminator of Cauliflower mosaic virus. Three independent transgenic lines were obtained and all of them exhibited strong systemic resistance to the simultaneous infection of the three viruses. These results demonstrate the effectiveness of this very straight forward strategy for engineering RNAi-based virus resistance in a major crop plant. More importantly, our strategy of construct assembly makes it easy to incorporate additional short IRs in the transgene, thus expanding the spectrum of virus resistance. Finally, this strategy could be easily adapted to control virus problems of other crop plants.     

First report of celery stunt anthracnose caused by <Emphasis Type="Italic">Colletotrichum simmondsii</Emphasis> in Japan

A new disease was found in Japan, on celery (Apium graveolens var. dulce) having severe chlorotic leaf spot, stunt, and dwarf with leaf curl. A spore suspension from the fungus isolated from affected plants induced identical symptoms 14 days after plants were sprayed. Identification and molecular characterization showed that the causal agent is Colletotrichum simmondsii. This report is the first of stunt anthracnose on celery caused by C. simmondsii. We propose the name “stunt anthracnose” for the new disease. Colletotrichum acutatum sensu lato, as reference pathogen of celery anthracnose, should be changed to C. fioriniae based on morphological and molecular characteristics.     

Plectosporium blight of monkshood caused by <Emphasis Type="Italic">Plectosporium tabacinum</Emphasis>

Severe blight of potted seedlings of monkshood caused by Plectosporium tabacinum was found in glasshouses in Kagawa Prefecture in southwest Japan in May 2001. Root rot and browning of stem bases were followed by wilting and yellowing of leaves, then blighting of leaves, flower buds and stems. A fungus was isolated from diseased plants and confirmed to cause the disease. The new disease was named “Plectosporium blight of monkshood”.     

Methane emissions from stems of Fraxinus mandshurica var. japonica trees in a floodplain forest

We measured methane (CH₄) emissions from the stem surfaces of mature Fraxinus mandshurica var. japonica trees in a floodplain forest. Flux measurements were conducted almost monthly from May to October 2005, and positive CH₄ fluxes were detected throughout the study period, including the leafless season. The mean CH₄ flux was 176 and 97 μg CH₄ m⁻² h⁻¹ at the lower (15 cm above the ground) and upper (70 cm above the ground) stem positions, respectively. The CH₄ concentration was lower in soil gas than in ambient air to a depth of at least 40 cm. One possible source of CH₄ emitted from the stems might be the dissolved CH₄ in groundwater; maximum concentrations were 10,000 times higher than atmospheric CH₄ concentrations. Our results suggest that CH₄ transport from the submerged soil layer to the atmosphere may occur through internal air spaces in tree bodies.     

Dual origin of the cultivated rice based on molecular markers of newly collected annual and perennial strains of wild rice species, Oryza nivara and O. rufipogon

The direct ancestor of rice (Oryza sativa L.) is believed to be AA genome wild relatives of rice in Asia. However, the AA genome wild relatives involve both annual and perennial forms. The distribution of the retrotransposon p-SINE1-r2, a short interspersed nuclear element (SINE) at the waxy locus was analyzed in diverse accessions of the AA genome wild relatives of rice (O. rufipogon sensu lato). Most annual wild rice accessions had this retrotransposon, while most perennial types lacked this element, contradicting results to the previous studies. Results presented here suggest that O. sativa has dual origin that lead to indica-japonica differentiation. Results suggest the indica line of rice varieties evolved from the annual genepool of AA genome and the japonica varieties from the perennial genepool of AA genome wild rice.     

2,4-Nonadienal and benzaldehyde bioantimutagens in Fushimi sweet pepper (Fushimi-togarashi)

Fushimi sweet pepper, "Fushimi-togarashi", is one of the "Kyo-yasai", traditional vegetables, in Kyoto, Japan. The chloroform fraction of Fushimi sweet pepper showed bioantimutagenicity on UV induced mutation in Escherichia coli B/r WP2. The bioantimutagen was purified with silica gel chromatography and identified as 2, 4-nonadienal (ID(50) = 20 microg/plate) on the basis of GC retention time and EI-MS spectrum of authentic 2,4-nonadienal. The sweet pepper also contained a known bioantimutagen, benzaldehyde (ID(50) = 2 mg/plate). Additive bioantimutagenicity was also observed by 2, 4-nonadienal with benzaldehyde. 2,4-Nonadienal did not show bioantimutagenicity in an UV excision repair deficient strain, E. coli B/r WP2s uvrA(-)(). Furthermore no delay of the first cell division after UV irradiation was observed in E. coli B/r WP2. These results indicate that the bioantimutagenic activity of 2, 4-nonadienal on UV mutagenesis might depend on the excision repair system in E. coli B/r WP2.     

Characteristics of fertilizer uptake and biodistribution in strawberry plants in two Japanese cultivars in hydroponic culture

ABSTRACT

The fertilizer absorption characteristics of strawberries are not clear, although appropriate fertilization is definitely necessary to ensure produce quality and quantity. This study aimed to determine the amounts of macro- and micronutrients absorbed during cultivation of strawberries and their biodistribution and utilization in the plant body. We cultivated Japanese strawberries ‘Benihoppe’ and ‘Kirapika’ in small hydroponic equipment containing a nutrient solution and determined the amounts of N, P, K, Ca, Mg, Fe, Mn, B, Zn, Cu, and Mo absorbed during and at the end of cultivation. The results revealed the adsorption levels of these elements during the cultivation period. The nutrient concentrations varied greatly among plant organs. In particular, P and B accumulated at high levels in the leaves and stem, K, Ca, Mg, Mn, Zn, and Cu accumulated in the crown, and N, Fe, and Mo accumulated in the roots. In addition, the uptake levels of N, P, K, Mg, Mn, Zn, and Cu differed between Benihoppe and Kirapika. Our results provide useful information for determining fertilizer application rates in strawberry cultivation.     

Analysis of strigolactones,germination stimulants for striga and orobanche,by high-performance liquid chromatography/tandem mass spectrometry

A simple and rapid analytical method for strigolactones, germination stimulants for the root parasitic weeds witchweed (Striga spp.) and broomrape (Orobanche spp.), has been developed using high-performance liquid chromatography connected to tandem mass spectrometry (LC/MS/MS). The natural strigolactones (strigol, sorgolactone, orobanchol, and alectrol) were clearly separated and identified by LC/MS/MS. As low as 0.1 pg/microL of strigol and 0.5 pg/microL of sorgolactone could be quantified, whereas 1 pg/microL was needed for the quantification of orobanchol (S/N > 10). Using this method, it was found that red clover produces orobanchol and alectrol but not strigol. The roots of red clover seedlings were found to produce 13, 70, 58, and 65 pg of orobanchol/plant 1, 2, 3, and 4 weeks after germination, respectively.     

Acidity neutralization mechanism in a forested watershed in Central Japan

The contributions of cation exchange and mineral weathering to the neutralization of acidity in the Jingahata watershed in central Japan were estimated through a laboratory weathering experiment and runoff chemistry measurements. The laboratory experiment was conducted in a stirred-flow reactor for a whole soil sample collected from the C horizon in the watershed. The concentration ratios of base cations (Ca²⁺, Mg²⁺, K⁺ and Na⁺) to Si (BC/Si) released during the steady-state stage of the laboratory experiment were in good agreement with the ratios of the net flux of base cations to the flux of Si in the streamwater (BC _{N ET}/Si _L).This result suggests that the acidity in the watershed is neutralized primarily by mineral weathering without causing a net loss of base cations from exchange sites. The alkalinity/acidity balance estimated for the watershed shows that the total weathering rate of base cations is approximately 3.26 keq ha^?1 yr^?1. Weathering of plagioclase (An₄₁) contributes 83% of the total weathering rate. The dominant acidity source is CO₂ released within the soil horizons, accounting for roughly 85% of the total acidity flux (3.20 keq ha^?1 yr^?1). This high internal production of acidity suppresses the relative importance of atmospheric acidity inputs (0.3 keq ha^?1 yr^?1).     

Comparison of Ni/Zn Accumulation Ability of Thlaspi japonicum from Three Different Areas in Hokkaido

Thlaspi japonicum is a Japanese Ni-hyperaccumulator plant growing in areas with serpentine soil containing a high level of Ni. In the present study, we investigated the ability of wild Thlaspi japonicum plants inhabiting non-ultramafic soil areas of Hokkaido to accumulate Ni/Zn. Wild T. japonicum plants growing on Rebun Island (Rebun-T) accumulated small amounts of Ni, while the plants cultivated on ultramafic rock soil accumulated large amounts of Ni, like T. japonicum growing on Mt. Yubari (Yubari-T), and this change was also observed for T. japonicum growing on Mt. Tengu (Tengu-T). All the T. japonicum plants transplanted on both ultramaflc soil or Andosol accumulated over 100 mg kg⁻¹ Zn, especially the Ni/Zn content of Tengu-T plants tended to be higher than that of the plants growing in the other two areas. In contrast, transplanting of Yubari-T plants from ultramafic soil with a high level of Ni to soil with a lower-Ni content decrease of the Ni concentration in the leaves. Wild Rebun-T plants showed a smaller biomass than the Yubari-T and Tengu-T plants, and some individuals of the Rebun-T population were smaller than those of the other populations, with tiny radical leaves. Zinc content of the small leaves was not significantly different from that in large leaves. These results suggested that T. japonicum plants are able to accumulate high Ni/Zn accumulation, regardless ecotype, habitat and leaf size.